1. Technical Field
The present invention relates to a mutant microorganism having the ability to produce a high concentration of putrescine and a method for producing putrescine using the same. More particularly, the present invention relates to a mutant microorganism having the ability to produce a high concentration of putrescine wherein gene(s) involved in the putrescine degradation or utilization pathway is inactivated or deleted, and a method for producing putrescine in high yield by culturing the microorganism.
2. Related Art
Putrescine (also known as 1,4-butanediamine), an important raw material for the production of polyamide-4,6, including nylon-4,6, is mainly produced on industrial scale by the hydrogenation of succinonitrile which is produced into acrylonitrile by addition of hydrogen cyanide. Known processes for the chemical synthesis of this compound require non-renewable petrochemical products as raw materials, and relatively severe reaction conditions of temperature and pressure in a multi-step and multi-reactor design, as well as the use of expensive catalyst systems. Furthermore, because these raw materials are highly toxic and flammable, the known chemical synthetic processes are environmentally disadvantageous. Accordingly, as an alternative to the chemical production process, a process of producing putrescine from a renewable biomass-derived carbon source is required.
Putrescine is a kind of polyamine which is found in a broad spectrum of organisms ranging from bacteria to animals and plants. For example, putrescine is known to play an important role not only in cell proliferation and normal cell growth, but also in a defensive mechanism against oxidative stress (Tkachenko et al., Arch. Microbiol., 176:155-157, 2001). Meanwhile, the intracellular levels of polyamines are strictly controlled by their biosynthesis, degradation, uptake, and secretion (Igarashi and Kashiwagi et al., J. Bacteriol., 170(7):3131-3135, 1988). The concentration of putrescine in E. coli is known to be as extremely high as about 2.8 g/l. Also, microorganisms have potentially good resistance to high concentrations of polyamines. For example, Mimitsuka et al. have reported that Corynebacterium glutamicum can grow even in the presence of more than 30 g/L of cadaverine. Accordingly, studies on the production of high-concentration polyamines (putrescine) using microorganisms have been continued.
European Patent Publication No. 0726240 A1 discloses a method of producing putrescine through fermentation using inexpensive industrial waste products or materials having protein as a major component. However, because the disclosed materials are very complex, there is a problem in that many purification steps have to be carried out in order to obtain putrescine and cadaverine. In addition, European Patent Publication No. 1784496 A1 discloses a process of biochemically synthesizing putrescine by microbial growth in a minimal salt medium containing glucose as a carbon source. According to this patent document, in order to improve the conversion of ornithine to putrescine, the activity of ornithine decarboxylase is increased by overexpression of an ornithine decarboxylase-encoding speC or speF. However, when the putrescine content is increased as a result of increasing ornithine decarboxylase, there are problems in that putrescine biosynthesis is inhibited and the degradation of putrescine is induced (Igarashi and Kashiwagi et al., Biochem. J., 347:297-303, 2000).
Studies on the degradation and utilization of putrescine in microorganisms are as follows. Bowman et al. have reported that spermidine synthase which is the product of the speE gene promotes the biosynthesis of spermidine from putrescine in E. coli (Bowman et al., J. Biol. Chem., 248:2480-2486, 1973). Spermidine synthase (EC:2.5.1.16) is present in most cell systems for the synthesis of spermidine.
Haywood et al. have reported that the yeast Candida boidinii induces the acetylation of putrescine to N-acetylputrescine in the presence of N-acetyltransferase. Spermidine acetyltransferase which is an E. coli speG gene product has high homology with the N-acetyltransferase of the yeast, and thus, must possess putrescine acetyltransferase (Haywood and Large, Eur. J. Biochem., 148:277-283, 1985).
Furthermore, Samsonova et al. have reported another putrescine degradation pathway in which a coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase without γ-glutamylation results in conversion of putrescine into γ-aminobutyric acid (Samsonova et al., BMC Microbiol., 3:2, 2003; Samsonova et al., FEBS Lett., 579:4107-4112, 2005).
Moreover, Kurihara et al. have called the putrescine degradation pathway as “Puu catabolic pathway” based on the findings that the putrescine degradation pathway is closely associated with γ-glutamylated metabolites of E. coli. Through such γ-glutamylation, γ-aminobutyraldehyde which is an aldehyde intermediate can be stabilized. The first reaction of this pathway is promoted by converting putrescine to γ-glutamyl-L-putrescine in the presence of γ-glutamylputrescine synthetase which is the product of the puuA gene. Also, it has been found that the catabolic pathway is a major factor for culturing E. coli in a medium containing putrescine as a sole nitrogen source. In addition, it has been found that a putrescine importer which is the product of the puuP gene is associated with the catabolic pathway and main putrescine importers (Kurihara et al., J. Biol. Chem., 280:4602-4608, 2005).
Accordingly, the present inventors have prepared mutant microorganisms wherein at least one gene selected from a speE gene encoding spermidine synthase, a speG gene encoding spermidine N-acetyltransferase, an argI gene encoding ornithine carbamoyltransferase chain I-monomer and a puuP gene encoding putrescine importer, which are involved in the putrescine degradation or utilization pathway of putrescine-producing microorganisms, is inactivated or deleted, and have found that, when the mutant microorganisms are cultured, they can produce a high concentration of putrescine, thereby completing the present invention.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.